Apoptosis, or programmed cell death, is a prominent feature of the nervous system during normal development and in adult brain exposed to environmental stress (Kuida et al., Nature, 384:368-372, 1996; Ratan et al., Neurochem., 62:376-379, 1994; Raff et al., Science, 262:695-700, 1993). Stress-induced apoptosis has been implicated in a variety of neurological diseases (Thompson, Science, 267:1456-1462, 1995) and requires de novo protein and RNA synthesis (Martin et al., J. Cell. Biol., 106:829-844, 1988; Oppenheim et al., Dev. Biol., 138:104-113, 1990). Increased expression of c-Jun protein is associated with neuronal damage following global ischemia (Neumann-Haefelin et al., Cerebral Flow Metab,, 14:206-216, 1994) or transection of nerve axons in vivo (Neumann-Haefelin, supra). Increased expression and phosphorylation of c-Jun have been observed in vitro prior to the apoptotic death of sympathetic neurons deprived of nerve growth factor (NGF) (Ham et al., Neuron, 14:927-939, 1995). Moreover, expression of a dominant negative mutant c-Jun, or treatment with c-Jun antibody protects NGF-deprived sympathetic neurons from apoptosis (Ham et al., supra; Estus et al., J. Cell. Biol., 127:1717-1727, 1994). However, the requirement of c-Jun for stress-induced neuronal apoptosis has not been tested in vivo since c-Jun deficient mice die during mid-gestation (Hilberg et al., Nature, 365:179-181, 1993).
Protein phosphorylation is one important mechanism involved in the activation of c-Jun in response to environmental stress signals (Whitmarsh et al., J. Mol. Med., 74:589-607, 1996). c-Jun N-terminal kinase (JNK, also known as SAPK) is a serine/threonine protein kinase that phosphorylates two residues (Ser-63 and Ser-73) on the NH2-terminal activation domain of c-Jun (Whitmarsh et al., supra; Dèrijard et al., Cell, 76:1025-1037, 1994; Kyriakis et al., Nature, 369:156-160, 1994). Map kinase kinase (MKK) 4 (also known as SEK1) is a direct activator of JNK in response to environmental stresses and mitogenic factors (Whitmarsh et al, supra; Dèrijard et al, supra; Nishina et al., Nature, 385:350-353, 1997; Yang et al., Proc. Nat. Acad. Sci. USA, 94:3004-3009, 1997; Sanchez et al., Nature, 372:794-798, 1994). JNK also phosphorylates ATF2 and other Jun-family proteins which function as components of the AP-1 transcription factor complex (Whitmarsh et al., supra; Gupta et al., Science, 267:389-393, 1995; Gupta et al., EMBO J., 15:2760-2770, 1996). The phosphorylation of these transcription factors by JNK leads to increased AP-1 transcriptional activity (Whitmarsh et al., supra). Conversely, the induction of AP-1 transcriptional activity is selectively blocked in cells lacking MKK4 (Yang et al., supra).
JNK has been implicated in the apoptosis of NGF-differentiated PC12 pheochromocytoma cells (Xia et al., Science, 270:1326-1311, 1995), one model system of neuronal cell death in vivo (Batistatou et al., J. Cell. Biol., 122:523-532, 1993). When differentiated PC12 cells are deprived of nerve growth factor (NGF), JNK activation is observed prior to apoptotic death (Xia et al., supra). Transfection studies using constitutively activated and dominant negative mutant components of the JNK signaling pathway established that JNK is involved in NGF withdrawal-induced apoptosis of PC12 cells (Xia et al., supra).
Ten JNK isoforms, resulting from alternative splicing of three different genes have been identified (D erijard et al., supra; Kyriakis et al., supra; Gupta et al., supra; Martin et al., Brain Res. Mol. Brain Res., 35:47-57, 1996). Although the JNK1 and JNK2 isoforms are widely expressed in murine tissues, including the brain, the JNK3 isoforms are predominantly expressed in the brain and, to a lesser extent, in the heart and testis.